DESCRIPTION (Taken directly from the application) The effectiveness of gene therapy approaches for cystic fibrosis will only be fully realized once a basic understanding of vector interactions with the target cell of the airway are understood. Ultimately, the best therapeutic approaches for gene therapy of CF will utilize single dose delivery of integrating vectors prior to the onset of disease. To date, only two vector systems including recombinant retroviruses and AAV have the ability to integrate recombinant transgenes within the host genomes. Retroviruses although extremely efficient in their integrating process are limited by achievable titers for in vivo delivery. In contrast AAV vectors provide a more attractive alternative with much higher achievable titers but have recently been troubled by the inefficiency of integration as studied in cell lines. However, the efficiency of the AAV; integrating process appears to be effected by the recipient cell type and cellular factors which regulate the integration process. Therefore, since the relative utility of AAV for gene therapy to the airway will be dependent on the recipient cell type, an obvious question remains as to the efficiency of integration in appropriate target progenitor cells of the human airway. This question is much different than the efficiency of AAV transduction in adult animal airways and hence must use alternative experimental approaches to investigate this question. Furthermore, the foundation of studies on wild type AAV suggests that the AAV recombinant genome integrates site specifically within chromosome 19. Although present studies evaluating the site specific integration of recombinant AAV in cell lines suggest that integration is random, little is known about the sequence specificity of this integration process. Such information could be used to potentially improve the efficiency and targeting of AAV integration. Using retroviral marking studies, our laboratory have defined multiple human airway progenitor cell targets for gene therapy with varying capacities for differentiation. Among these progenitors is included a punitive stem cell which has pluripotent development for surface airway epithelium as well as submucosal liands. The most functionally relevant cellular targets for gene therapy involve this stem cell or alternatively those progenitor cells which give rise to CFTR expressing cells in the airway. To evaluate the utility of AAV vectors for in utero gene therapy in the airway and submucosal glands, we propose to evaluate MV integration in progenitor cell subsets using a bronchial xenograft model system. Studies evaluating AAV integration in cell lines demonstrate a high level of variability in the efficiency of integration. These differences may be due to cell specific variability in the expression of cellular factors which influence this integration process. To this end, we hypothesize that the efficiency of integration in airway progenitor cells may also vary based on the differences in biology of these target cells as indicated by their varying capacity for differentiation and regeneration. Furthermore, we propose to developed an Alu PCR method by which integration sites of AAV can be cloned from in vivo microdissected progenitor cell clones. This information will allow for direct evaluation of integration sequence specificity and may ultimately provide a better understand of the cellular differences which effect the integration process. A better understanding of the interactions of AAV vectors with the appropriate airway target progenitor cells may ultimately lead to rational methods to alter vector integration efficiencies and site specificities.